Transient transfection method for retroviral production

ABSTRACT

The invention relates to bacterial artificial chromosomes (BAC) comprising retroviral nucleic acid sequences encoding: gag and pol proteins, and an env protein or a functional substitute thereof, wherein each of the retroviral nucleic acid sequences are arranged as individual expression constructs within the BAC. The invention also relates to uses and methods of transient transfection using said BAC.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/356,813 filed on 21 Nov. 2016, which claims the benefit of U.K.Provisional Application No. GB 1520764.0, filed 24 Nov. 2015 and GB1609354.4, filed 26 May 2016.

FIELD OF THE INVENTION

The invention relates to nucleic acid vectors, in particular bacterialartificial chromosomes, comprising genes required for retroviralproduction and uses thereof. Also provided are methods of producingreplication defective retroviral vector particles comprising the nucleicacid vectors as described herein.

BACKGROUND TO THE INVENTION

In gene therapy, genetic material is delivered to endogenous cells in asubject in need of treatment. The genetic material may introduce novelgenes to the subject, or introduce additional copies of pre-existinggenes, or introduce different alleles or variants of genes that arepresent in the subject. Viral vector systems have been proposed as aneffective gene delivery method for use in gene therapy (Verma and Somia(1997) Nature 389: 239-242).

In particular, these viral vectors are based on members of theretrovirus family due to their ability to integrate their geneticpayload into the host's genome. Retroviral vectors are designed to keepthe essential proteins required for packaging and delivery of theretroviral genome, but any non-essential accessory proteins includingthose responsible for their disease profile are removed. Examples ofretroviral vectors include lentiviral vectors, such as those based uponHuman Immunodeficiency Virus Type 1 (HIV-1), which are widely usedbecause they are able to integrate into non-proliferating cells.

Currently, the majority of viral vectors are produced by transientco-transfection of viral genes into a host cell line. The viral genesare introduced using bacterial plasmids which exist in the host cell foronly a limited period of time because the viral genes remain on theplasmids and are not integrated into the genome. As such, transientlytransfected genetic material is not passed on to subsequent generationsduring cell division.

However, there have been several problems associated with the methods oftransient transfection currently used, such as batch-to-batchvariability, the high cost of transfection reagents and the difficultyto maintain quality control (see Segura et al. (2013) Expert Opin. Biol.Ther. 13(7): 987-1011). The process of transfection itself is alsolabour-intensive and challenging to scale up. There is also thedifficult task of removing plasmid impurities which are carried overduring vector preparation (see Pichlmair et al. (2007) J. Virol. 81(2):539-47).

It is therefore an object of the present invention to provide animproved method of transient transfection which overcomes one or more ofthe disadvantages associated with existing methods.

SUMMARY OF THE INVENTION

The present inventors have developed a new way of producing retroviralvectors which involves the use of nucleic acid vectors comprising anon-mammalian origin of replication and the ability to hold at least 25kilobases (kb) of DNA, such as bacterial artificial chromosomes,comprising all of the retroviral genes essential for retroviral vectorproduction. Current methods of transient transfection require the use of3-4 separate plasmids carrying different components required forretroviral production to be introduced into the host cell which is timeconsuming and causes problems associated with selection pressure. Themethod proposed by the present inventors incorporates all of theessential retroviral genes on a single construct which can then beintroduced into a host cell which reduces the amount of materialrequired to transduce the host cell for viral vector production.Therefore, this reduces the cost of goods because only a single vectoris used, rather than previous methods which use multiple plasm idvectors.

The use of a nucleic acid vector comprising a non-mammalian origin ofreplication and which has the ability to hold at least 25 kb of DNA(i.e. large-construct DNA) has several advantages. In the firstinstance, the vectors can first be manipulated in non-mammalian cells(e.g. microbial cells, such as bacterial cells) rather than mammalianhost cells which makes them much easier to use (e.g. bacterialartificial chromosomes can first be manipulated in E. coli). Once thenucleic acid vector has been prepared, it can be introduced into a hostcell, such as a mammalian host cell, for retroviral vector production.

The use of nucleic acid vectors of the present invention thereforeprovides advantages in the generation of retroviral vectors.

Therefore, according to a first aspect of the invention, there isprovided a bacterial artificial chromosome (BAC), characterized in thatsaid BAC comprises retroviral nucleic acid sequences encoding:

gag and pol proteins, and

an env protein or a functional substitute thereof

wherein each of the retroviral nucleic acid sequences are arranged asindividual expression constructs within the BAC.

According to a further aspect of the invention, there is provided theBAC defined herein for use in a method of producing retroviral vectorparticles.

According to a further aspect of the invention, there is provided amethod of producing a replication defective retroviral vector particle,comprising:

(a) introducing the BAC as defined herein into a culture of mammalianhost cells; and

(b) culturing the mammalian host cells under conditions in which thereplication defective retroviral vector particle is produced.

According to a further aspect of the invention, there is provided areplication defective retroviral vector particle obtained by the methodas defined herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A stepwise guide to the construction of BACpack-WTGP-277delU5and BACpack-SYNGP-277delU5.

FIG. 2: Comparison of viral titres obtained in Example 2. 10⁶ HEK293Tcells were seeded in a 6 well plate. The following day, the adheredcells were transfected using PEI to manufacturer's instruction. Cellswere either transfected with a total 4 μg of Wild Type (WT) lentiviralpackaging constructs consisting of pMDL.gp (GagPol), pMD.G (VSVg),pK-Rev (Rev) and pCCL.277 (GFP Transfer vector) or 2 μg BACpack (asingle BAC construct containing GagPol, VSVg and Rev) plus 2 μg of theeGFP transfer vector on a seperate plasmid.

48 hours post transfection, the supernatant was harvested, filteredthrough a 0.22 μm filter and stored at −80° C. for a minimum of 4 hours.HEK293T cells were seeded for transduction at 10⁵ cells per well in a 24well plate. The following day viral supernatant was applied to the cellsin serial dilutions with Polybrene at a final concentration of 8 μg/ml.3 days post transduction the cells were harvested by trypsin treatmentand analysed for GFP by FACS. Viral titre was calculated as TransductionUnits (TU)/mL using the following equation:

(GFP positive cells/100)×dilution factor×number of cells transduced.

Viral titres are compared on the bar chart. All incubations were carriedout at 37° C. and 5% CO₂. Media used was DMEM supplemented with FBS to10% and 1 μg/ml Doxycycline in the BACpack+Transfer sample.

FIG. 3: Transient Transfection of the BACpack in Adherent HEK293T cells.HEK293T cells transiently transfected with BACpackWTGP-277delU5,BACpackSYN-277delU5 or the standard 4 plasmid system using the CalciumPhosphate method. 16 hours post transfection, the +Dox conditions wereinduced for 24 hours with 1 μg/ml doxycycline. Viral supernatant washarvested 48 post transfection, filtered through a 0.22 μm filter andtitrated by transducing HEK293T cells. GFP positive transduced cellswere used to calculate the Transducing Units/ml (TU/ml).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entirety.

The term “comprising” encompasses “including” or “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

The term “consisting essentially of” limits the scope of the feature tothe specified materials or steps and those that do not materially affectthe basic characteristic(s) of the claimed feature.

The term “consisting of” excludes the presence of any additionalcomponent(s).

The term “about” in relation to a numerical value x means, for example,x±10%, 5%, 2% or 1%.

The term “vector” or “nucleic acid vector” refers to a vehicle which isable to artificially carry foreign (i.e. exogenous) genetic materialinto another cell, where it can be replicated and/or expressed. Examplesof vectors include non-mammalian nucleic acid vectors, such as bacterialartificial chromosomes (BACs), yeast artificial chromosomes (YACs),P1-derived artificial chromosomes (PACs), cosmids or fosmids.

Other examples of vectors include viral vectors, such as retroviral andlentiviral vectors, which are of particular interest in the presentapplication. Lentiviral vectors, such as those based upon HumanImmunodeficiency Virus Type 1 (HIV-1) are widely used as they are ableto integrate into non-proliferating cells. Viral vectors can be madereplication defective by splitting the viral genome into separate parts,e.g., by placing on separate plasmids. For example, the so-called firstgeneration of lentiviral vectors, developed by the Salk Institute forBiological Studies, was built as a three-plasmid expression systemconsisting of a packaging expression cassette, the envelope expressioncassette and the vector expression cassette. The “packaging plasmid”contains the entire gag-pol sequences, the regulatory (tat and rev) andthe accessory (vif, vpr, vpu, net) sequences. The “envelope plasmid”holds the Vesicular stomatitis virus glycoprotein (VSVg) in substitutionfor the native HIV-1 envelope protein, under the control of acytomegalovirus (CMV) promoter. The third plasmid (the “transferplasmid”) carries the Long Terminal Repeats (LTRs), encapsulationsequence (y), the Rev Response Element (RRE) sequence and the CMVpromoter to express the transgene inside the host cell.

The second lentiviral vector generation was characterized by thedeletion of the virulence sequences vpr, vif, vpu and nef. The packagingvector was reduced to gag, pol, tat and rev genes, therefore increasingthe safety of the system.

To improve the lentiviral system, the third-generation vectors have beendesigned by removing the tat gene from the packaging construct andinactivating the LTR from the vector cassette, therefore reducingproblems related to insertional mutagenesis effects.

The various lentivirus generations are described in the followingreferences: First generation: Naldini et al. (1996) Science 272(5259):263-7; Second generation: Zufferey et al. (1997) Nat. Biotechnol. 15(9):871-5; Third generation: Dull et al. (1998) J. Virol. 72(11): 8463-7,all of which are incorporated herein by reference in their entirety. Areview on the development of lentiviral vectors can be found in Sakumaet al. (2012) Biochem. J. 443(3): 603-18 and Picango-Castro et al.(2008) Exp. Opin. Therap. Patents 18(5):525-539.

The term “non-mammalian origin of replication” refers to a nucleic acidsequence where replication is initiated and which is derived from anon-mammalian source. This enables the nucleic acid vectors of theinvention to stably replicate and segregate alongside endogenouschromosomes in a suitable host cell (e.g. a microbial cell, such as abacterial or yeast cell) so that it is transmittable to host cellprogeny, except when the host cell is a mammalian host cell. Inmammalian host cells, nucleic acid vectors with non-mammalian origins ofreplication will either integrate into the endogenous chromosomes of themammalian host cell or be lost upon mammalian host cell replication. Forexample, nucleic acid vectors with non-mammalian origins of replicationsuch as bacterial artificial chromosomes (BAC), P1-derived artificialchromosome (PAC), cosmids or fosmids, are able to stably replicate andsegregate alongside endogenous chromosomes in bacterial cells (such asE. coli), however if they are introduced into mammalian host cells, theBAC, PAC, cosmid or fosmid will either integrate or be lost uponmammalian host cell replication. Yeast artificial chromosomes (YAC) areable to stably replicate and segregate alongside endogenous chromosomesin yeast cells, however if they are introduced into mammalian hostcells, the YAC will either integrate or be lost upon mammalian host cellreplication. Therefore, in this context, the nucleic acid vectors of theinvention act as reservoirs of DNA (i.e. for the genes essential forretroviral production) which can be easily transferred into mammaliancells to generate retroviral vector particles. Examples of non-mammalianorigins of replication include: bacterial origins of replications, suchas oriC, oriV or oriS; viral origins of replication, such as SV40 originof replication; or yeast origins of replication, also known asAutonomously Replicating Sequences (ARS elements).

The nucleic acid vectors of the present invention comprise anon-mammalian origin of replication and are able to hold at least 25kilobases (kb) of DNA. In one embodiment, the nucleic acid vector hasthe ability to hold at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350kb of DNA. It will be understood that references to “ability to hold”has its usual meaning and implies that the upper limit for the size ofinsert for the nucleic acid vector is not less than the claimed size(i.e. not less than 25 kb of DNA).

The aim of the present invention is to include the genes essential forretroviral packaging in a single construct (i.e. the nucleic acidvector). Therefore, the nucleic acid vectors of the invention, must beable to hold large inserts of DNA. For the avoidance of doubt, it willbe understood that references to “nucleic acid vectors” or “artificialchromosomes” do not refer to natural bacterial plasmids (e.g. such asthe plasmids currently used in transient transfection methods) becausethese are not able to hold at least 25 kb of DNA. The maximum sizeinsert which a plasmid can contain is about 15 kb. Such nucleic acidvectors also do not refer to bacteriophages which generally only holdmaximum inserts of 5-11 kb. Therefore, in one embodiment the nucleicacid vector of the invention is not a plasmid, bacteriophage or episome.

The term “endogenous chromosomes” refers to genomic chromosomes found inthe host cell prior to generation or introduction of an exogenousnucleic acid vector, such as a bacterial artificial chromosome.

The terms “transfection”, “transformation” and “transduction” as usedherein, may be used to describe the insertion of the non-mammalian orviral vector into a target cell. Insertion of a vector is usually calledtransformation for bacterial cells and transfection for eukaryoticcells, although insertion of a viral vector may also be calledtransduction. The skilled person will be aware of the differentnon-viral transfection methods commonly used, which include, but are notlimited to, the use of physical methods (e.g. electroporation, cellsqueezing, sonoporation, optical transfection, protoplast fusion,impalefection, magnetofection, gene gun or particle bombardment),chemical reagents (e.g. calcium phosphate, highly branched organiccompounds or cationic polymers) or cationic lipids (e.g. lipofection).Many transfection methods require the contact of solutions of plasmidDNA to the cells, which are then grown.

The term “promoter” refers to a sequence that drives gene expression. Inorder to drive a high level of expression, it may be beneficial to use ahigh efficiency promoter, such as a non-retroviral, high efficiencypromoter. Examples of suitable promoters may include a promoter such asthe human cytomegalovirus (CMV) immediate early promoter, spleenfocus-forming virus (SFFV) promoter, Rous sarcoma virus (RSV) promoter,or human elongation factor 1-alpha (pEF) promoter.

The term “polyA signal” refers to a polyadenylation signal sequence, forexample placed 3′ of a transgene, which enables host factors to add apolyadenosine (polyA) tail to the end of the nascent mRNA duringtranscription. The polyA tail is a stretch of up to 300 adenosineribonucleotides which protects mRNA from enzymatic degradation and alsoaids in translation. Accordingly, the nucleic acid vectors of thepresent invention may include a polyA signal sequence such as the humanbeta globin or rabbit beta globin polyA signals, the simian virus 40(SV40) early or late polyA signals, the human insulin polyA signal, orthe bovine growth hormone polyA signal. In one embodiment, the polyAsignal sequence is the human beta globin polyA signal.

The term “intron sequence” refers to a nucleotide sequence which isremoved from the final gene product by RNA splicing. The use of anintron downstream of the enhancer/promoter region and upstream of thecDNA insert has been shown to increase the level of gene expression. Theincrease in expression depends on the particular cDNA insert.Accordingly, the nucleic acid vector of the present invention mayinclude introns such as human beta globin intron, rabbit beta globinintron II or a chimeric human beta globin-immunoglobulin intron. In oneembodiment, the intron is a human beta globin intron and/or a rabbitbeta globin intron II.

The term “packaging cell line” refers to a cell line which is capable ofexpressing gag and pol protein and envelope glycoprotein genes.Alternatively, the term “producer cell line” refers to a packaging cellline which is also capable of expressing a transfer vector containing atransgene of interest.

The term “transiently transfected” refers to transfected cells where thetarget nucleic acids (i.e. retroviral genes) are not permanentlyincorporated into the cellular genome. Therefore, the effects of thenucleic acids within the cell last only a short amount of time.

Nucleic Acid Vectors

According to one aspect of the invention, there is provided a nucleicacid vector comprising a non-mammalian origin of replication and theability to hold at least 25 kilobases (kb) of DNA, characterized in thatsaid nucleic acid vector comprises retroviral nucleic acid sequencesencoding:

gag and pol proteins, and

an env protein or a functional substitute thereof.

In particular, each of the retroviral nucleic acid sequences may bearranged as individual expression constructs within the nucleic acidvector.

The present inventors have found that nucleic acid vectors describedherein can be used to generate retroviral vector particles whichameliorate previous difficulties associated with retroviral vectorproduction methods. For example, by including all of the essentialretroviral genes in the nucleic acid vector, the retroviral genes canthen be introduced into a mammalian host cell in one single step.Therefore, the use of a nucleic acid vector, as proposed herein, allowsfor fast vector production and reduces the amount of material requiredfor retroviral vector production.

In one embodiment, the nucleic acid vector additionally comprisesnucleic acid sequences which encode the RNA genome of a retroviralvector particle. It will be understood that the RNA genome of theretroviral vector particle is usually included on the “transfer vector”used in transient transfection methods. The transfer vector plasmidgenerally contains a promoter (such as CMV), the 3′ LTR (which may ormay not be a self-inactivating (i.e. SIN) 3′-LTR), the 5′ LTR (which mayor may not contain the U5 region), the encapsidation sequence (y) andpotentially the transgene linked to a promoter.

In one embodiment, multiple copies of the RNA genome of the retroviralvector particle (i.e. the transfer vector) are included in the nucleicacid vector. Multiple copies of the transfer vector are expected toresult in higher viral vector titre. For example, the nucleic acidvector may include two or more, such as three, four, five, six, seven,eight, nine or ten or more copies of the RNA genome of the retroviralvector particle (i.e. the transfer vector).

In one embodiment, the nucleic acid vector contains one or a pluralityof recombination site(s). The recombinase enzyme catalyses therecombination reaction between two recombination sites.

Many types of site-specific recombination systems are known in the art,and any suitable recombination system may be used in the presentinvention. For example, in one embodiment the recombination site(s) areselected or derived from the int/attsystem of lambda phage, the Cre/loxsystem of bacteriophage P1, the FLP/FRT system of yeast, the Gin/gixrecombinase system of phage Mu, the Cin recombinase system, the Pinrecombinase system of E. coli and the R/RS system of the pSR1 plasmid,or any combination thereof. In a further embodiment, the recombinationsite is an att site (e.g. from lambda phage), wherein the att sitepermits site-directed integration in the presence of a lambda integrase.It will be understood that the reference to “lambda integrase” includesreferences to mutant integrases which are still compatible with theint/attsystem, for example the modified lambda integrases described inWO 2002/097059.

In one embodiment, the nucleic acid vector is selected from: a bacterialartificial chromosome (BAC), a yeast artificial chromosome (YAC), aP1-derived artificial chromosome (PAC), fosmid or a cosmid. In apreferred embodiment, the nucleic acid vector is a bacterial artificialchromosome (BAC).

Bacterial Artificial Chromosomes

According to one aspect of the invention, there is provided a bacterialartificial chromosome (BAC), characterized in that said BAC comprisesretroviral nucleic acid sequences encoding:

gag and pol proteins, and

an env protein or a functional substitute thereof

wherein each of the retroviral nucleic acid sequences are arranged asindividual expression constructs within the BAC.

The term “bacterial artificial chromosome” or “BAC” refers to a DNAconstruct derived from bacterial plasmids which is able to hold a largeinsert of exogenous DNA. They can usually hold a maximum DNA insert ofapproximately 350 kb. BACs were developed from the well characterisedbacterial functional fertility plasmid (F-plasmid) which containspartition genes that promote the even distribution of plasmids afterbacterial cell division. This allows the BACs to be stably replicatedand segregated alongside endogenous bacterial genomes (such as E. coli).The BAC usually contains at least one copy of an origin of replication(such as the oriS or oriV gene), the repE gene (for plasmid replicationand regulation of copy number) and partitioning genes (such as sopA,sopB, parA, parB and/or parC) which ensures stable maintenance of theBAC in bacterial cells. BACs are naturally circular and supercoiledwhich makes them easier to recover than linear artificial chromosomes,such as YACs. They can also be introduced into bacterial host cellsrelatively easily, using simple methods such as electroporation.

In one embodiment, the bacterial artificial chromosome comprises an oriSgene. In one embodiment, the bacterial artificial chromosome comprises arepE gene. In one embodiment, the bacterial artificial chromosomecomprises partitioning genes. In a further embodiment, the partitioninggenes are selected from sopA, sopB, parA, parB and/or parC. In a yetfurther embodiment, the bacterial artificial chromosome comprises a sopAand sopB gene.

BAC for use in the present invention may be obtained from commercialsources, for example the pSMART BAC from LUCIGEN™ (see Genome AccessionNo. EU101022.1 for the full back bone sequence). This BAC contains theL-arabinose “copy-up” system which also contains the oriV medium-copyorigin of replication, which is active only in the presence of the TrfAreplication protein. The gene for TrfA may be incorporated into thegenome of bacterial host cells under control of the L-arabinoseinducible promoter araC-Paan (see Wild et al. (2002) Genome Res. 12(9):1434-1444). Addition of L-arabinose induces expression of TrfA, whichactivates oriV, causing the plasmid to replicate to up to 50 copies percell.

Yeast Artificial Chromosomes

The term “yeast artificial chromosome” or “YAC” refers to chromosomes inwhich yeast DNA is incorporated into bacterial plasmids. They contain anautonomous replication sequence (ARS) (i.e. an origin of replication), acentromere and telomeres. Unlike BACs, the YAC is linear and thereforecontains yeast telomeres at each end of the chromosome to protect theends from degradation as it is passed onto host cell progeny. YACs canhold a range of DNA insert sizes; anything from 100-2000 kb.

P1-Derived Artificial Chromosomes

The term “P1-derived artificial chromosome” or “PAC” refers to DNAconstructs derived from the DNA of the P1-bacteriophage and bacterialF-plasmid. They can usually hold a maximum DNA insert of approximately100-300 kb and are used as cloning vectors in E. coli. PACs have similaradvantages as BACs, such as being easy to purify and introduce intobacterial host cells.

Cosmids and Fosmids

The term “cosmid” refers to DNA constructs derived from bacterialplasmids which additionally contain cos sites derived from bacteriophagelambda. Cosmids generally contain a bacterial origin of replication(such as oriV), a selection marker, a cloning site and at least one cossite. Cosmids can usually accept a maximum DNA insert of 40-45 kb.Cosmids have been shown to be more efficient at infecting E. coli cellsthan standard bacterial plasmids. The term “fosmids” refers tonon-mammalian nucleic acid vectors which are similar to cosmids, exceptthat they are based on the bacterial F-plasmid. In particular, they usethe F-plasmid origin of replication and partitioning mechanisms to allowcloning of large DNA fragments. Fosmids can usually accept a maximum DNAinsert of 40 kb.

Retroviruses

Retroviruses are a family of viruses which contain a pseudo-diploidsingle-stranded RNA genome. They encode a reverse transcriptase whichproduces DNA from the RNA genome which can then be inserted into thehost cell DNA. The invention described herein may be used to producereplication defective retroviral vector particles. The retroviral vectorparticle of the present invention may be selected from or derived fromany suitable retrovirus.

In one embodiment, the retroviral vector particle is derived from, orselected from, a lentivirus, alpha-retrovirus, gamma-retrovirus orfoamy-retrovirus, such as a lentivirus or gamma-retrovirus, inparticular a lentivirus. In a further embodiment, the retroviral vectorparticle is a lentivirus selected from the group consisting of HIV-1,HIV-2, SIV, FIV, EIAV and Visna. Lentiviruses are able to infectnon-dividing (i.e. quiescent) cells which makes them attractiveretroviral vectors for gene therapy. In a yet further embodiment, theretroviral vector particle is HIV-1 or is derived from HIV-1. Thegenomic structure of some retroviruses may be found in the art. Forexample, details on HIV-1 may be found from the NCBI Genbank (GenomeAccession No. AF033819). HIV-1 is one of the best understoodretroviruses and is therefore often used as a retroviral vector.

Retroviral Genes

The nucleic acid sequences common to all retroviruses may be explainedin more detail, as follows:

Long Terminal Repeats (LTRs): The basic structure of a retrovirus genomecomprises a 5′-LTR and a 3′-LTR, between or within which are located thegenes required for retroviral production. The LTRs are required forretroviral integration and transcription. They can also act as promotersequences to control the expression of the retroviral genes (i.e. theyare cis-acting genes). The LTRs are composed of three sub-regionsdesignated U3, R, U5: U3 is derived from the sequence unique to the 3′end of the RNA; R is derived from a sequence repeated at both ends ofthe RNA; and U5 is derived from the sequence unique to the 5′ end of theRNA. Therefore, in one embodiment, the nucleic acid vector additionallycomprises a 5′- and 3′-LTR. In a further embodiment, the U5 region ofthe 5′ LTR can be deleted and replaced with a non-HIV-1 polyA tail (seeHanawa et al. (2002) Mol. Ther. 5(3): 242-51).

In order to address safety concerns relating to the generation ofreplication-competent virus, a self-inactivating (SIN) vector has beendeveloped by deleting a section in the U3 region of the 3′ LTR, whichincludes the TATA box and binding sites for transcription factors Sp1and NF-κB (see Miyoshi et al. (1998) J. Virol 72(10):8150-7). Thedeletion is transferred to the 5′ LTR after reverse transcription andintegration in infected cells, which results in the transcriptionalinactivation of the LTR. This is known as a self-inactivatinglentiviral-based vector system which may be included in the presentinvention.

ψ: Encapsidation of the retroviral RNAs occurs by virtue of a ψ (psi)sequence located at the 5′ end of the retroviral genome. It is also wellknown in the art that sequences downstream of the psi sequence andextending into the gag coding region are involved in efficientretroviral vector production (see Cui et al. (1999) J. Virol. 73(7):6171-6176). In one embodiment, the nucleic acid vector additionallycomprises a ψ (psi) sequence.

Primer Binding Site (PBS): The retroviral genome contains a PBS which ispresent after the U5 region of the 5′-LTR. This site binds to the tRNAprimer required for initiation of reverse transcription. In oneembodiment, the nucleic acid vector additionally comprises a PBSsequence.

PPT: Retroviral genomes contain short stretches of purines, calledpolypurine tracts (PPTs), near the 3′ end of the retroviral genome.These PPTs function as RNA primers for plus-strand DNA synthesis duringreverse transcription. Complex retroviruses (such as HIV-1) contain asecond, more centrally located PPT (i.e. a central polypurine tract(cPPT)) that provides a second site for initiation of DNA synthesis.Retroviral vectors encoding a cPPT have been shown to have enhancedtransduction and transgene expression (see Barry et al. (2001) Hum. GeneTher. 12(9):1103-8). In one embodiment, the nucleic acid vectoradditionally comprises a 3′-PPT sequence and/or a cPPT sequence.

The genomic structure of the non-coding regions described above are wellknown to a person skilled in the art. For example, details on thegenomic structure of the non-coding regions in HIV-1 may be found fromthe NCBI Genbank with Genome Accession No. AF033819, or for HIV-1 HXB2(a commonly used HIV-1 reference strain) with Genome Accession No.K03455. In one embodiment, the non-coding regions are derived from thesequences available at Genome Accession No. K03455, for example frombase pairs 454-1126 (for R-U5-PBS-Gag), 7622-8479 (for RRE) or 7769-8146(for RRE), 4781-4898 (for cPPT), 9015-9120 & 9521-9719 (fordNEF-PPT-sinU3-R-U5).

Gag/pol:

The expression of gag and pol genes relies on a translational frameshiftbetween gag and gagpol. Both are polyproteins which are cleaved duringmaturation. The major structural matrix, capsid, and nucleocapsidproteins of the retroviral vector are encoded by gag. The pol gene codesfor the retroviral enzymes: i) reverse transcriptase, essential forreverse transcription of the retroviral RNA genome to double strandedDNA, ii) integrase, which enables the integration of the retroviral DNAgenome into a host cell chromosome, and iii) protease, that cleaves thesynthesized polyprotein in order to produce the mature and functionalproteins of the retrovirus. In one embodiment, the retroviral nucleicacid sequence encoding the gag and pol proteins is derived from theHIV-1 HXB2 sequence, which is available at Genome Accession No. K03455,for example from base pairs 790-5105.

Env:

The env (“envelope”) gene codes for the surface and transmembranecomponents of the retroviral envelope (e.g. glycoproteins gp120 and gp41of HIV-1) and is involved in retroviral-cell membrane fusion. In orderto broaden the retroviral vector's tissue tropism, the retroviralvectors described herein may be pseudotyped with an envelope proteinfrom another virus. Pseudotyping refers to the process whereby the hostcell range of retroviral vectors, including lentiviral vectors, can beexpanded or altered by changing the glycoproteins (GPs) on theretroviral vector particles (e.g. by using GPs obtained from or derivedfrom other enveloped viruses or using synthetic/artificial GPs). Themost commonly used glycoprotein for pseudotyping retroviral vectors isthe Vesicular stomatitis virus GP (VSVg), due to its broad tropism andhigh vector particle stability. However, it will be understood by theskilled person that other glycoproteins may be used for pseudotyping(see Cronin et al. (2005) Curr. Gene Ther. 5(4):387-398, hereinincorporated by reference in its entirety). The choice of virus used forpseudotyping may also depend on the type of cell and/or organ to betargeted because some pseudotypes have been shown to have tissue-typepreferences.

In one embodiment, the env protein or a functional substitute thereof isobtained from or derived from a virus selected from a Vesiculovirus(e.g. Vesicular stomatitis virus), Lyssavirus (e.g. Rabies virus, Mokolavirus), Arenavirus (e.g. Lymphocytic choriomeningitis virus (LCMV)),Alphavirus (e.g. Ross River virus (RRV), Sindbis virus, Semliki Forestvirus (SFV), Venezuelan equine encephalitis virus), Filovirus (e.g.Ebola virus Reston, Ebola virus Zaire, Lassa virus), Alpharetrovirus(e.g. Avian leukosis virus (ALV)), Betaretrovirus (e.g. Jaagsiekte sheepretrovirus (JSRV)), Gammaretrovirus (e.g. Moloney Murine leukaemia virus(MLV), Gibbon ape leukaemia virus (GALV), Feline endogenous retrovirus(RD114)), Deltaretrovirus (e.g. Human T-lymphotrophic virus 1 (HTLV-1)),Spumavirus (e.g. Human foamy virus), Lentivirus (e.g. Maedi-visna virus(MVV)), Coronavirus (e.g. SARS-CoV), Respirovirus (e.g. Sendai virus,Respiratory syncytia virus (RSV)), Hepacivirus (e.g. Hepatitis C virus(HCV)), Influenzavirus (e.g. Influenza virus A) and Nucleopolyhedrovirus(e.g. Autographa californica multiple nucleopolyhedrovirus (AcMNPV)). Ina further embodiment, the env protein or a functional substitute thereofis obtained from or derived from Vesicular stomatitis virus. In thisembodiment, the Vesicular stomatitis virus glycoprotein (VSVg) proteinmay be used which enables the retroviral particles to infect a broaderhost cell range and eliminates the chances of recombination to producewild-type envelope proteins. In a further embodiment, the retroviralnucleic acid sequence encoding the env protein or a functionalsubstitute thereof, is derived from the sequence available at GenomeAccession No. J02428.1, for example from base pairs 3071 to 4720.

The structural genes described herein are common to all retroviruses.Further auxiliary genes may be found in different types of retrovirus.For example, lentiviruses, such as HIV-1, contain six further auxiliarygenes known as rev, vif, vpu, vpr, nef and tat. Other retroviruses mayhave auxiliary genes which are analogous to the genes described herein,however they may not have always been given the same name as in theliterature. References such as Tomonaga and Mikami (1996) J. Gen. Virol.77(Pt 8):1611-1621 describe various retrovirus auxiliary genes.

Rev: The auxiliary gene rev (“regulator of virion”) encodes an accessoryprotein which binds to the Rev Response element (RRE) and facilitatesthe export of retroviral transcripts. The gene's protein product allowsfragments of retroviral mRNA that contain the Rev Responsive element(RRE) to be exported from the nucleus to the cytoplasm. The RRE sequenceis predicted to form a complex folded structure. This particular role ofrev reflects a tight coupling of the splicing and nuclear export steps.In one embodiment, nucleic acid vector comprises an RRE sequence. In afurther embodiment, the RRE sequence is derived from HIV-1 HXB2sequence, which is available at Genome Accession No. K03455, for examplefrom base pairs 7622 to 8479, or 7769 to 8146, in particular base pairs7622 to 8479.

Rev binds to RRE and facilitates the export of singly spliced (env, vif,vpr and vpu) or non-spliced (gag, pol and genomic RNA) viraltranscripts, thus leading to downstream events like gene translation andpackaging (see Suhasini and Reddy (2009) Curr. HIV Res. 7(1): 91-100).In one embodiment, the nucleic acid vector additionally comprises theauxiliary gene rev or an analogous gene thereto (i.e. from otherretroviruses or a functionally analogous system). Inclusion of the revgene ensures efficient export of RNA transcripts of the retroviralvector genome from the nucleus to the cytoplasm, especially if an RREelement is also included on the transcript to be transported. In afurther embodiment, the rev gene comprises at least 60% sequenceidentity, such as at least 70% sequence identity to base pairs 970 to1320 of Genome Accession No. M11840 (i.e. HIV-1 clone 12 cDNA, theHIVPCV12 locus). In an alternative embodiment, the rev gene comprises atleast 60% sequence identity, such as at least 70%, 80%, 90% or 100%sequence identity to base pairs 5970 to 6040 and 8379 to 8653 of GenomeAccession No. K03455.1 (i.e. Human immunodeficiency virus type 1, HXB2).

Auxiliary genes are thought to play a role in retroviral replication andpathogenesis, therefore many current viral vector production systems donot include some of these genes. The exception is rev which is usuallypresent or a system analogous to the rev/RRE system is potentially used.Therefore, in one embodiment, the nucleic acid sequences encoding one ormore of the auxiliary genes vpr, vif, vpu, tat and nef, or analogousauxiliary genes, are disrupted such that said auxiliary genes areremoved from the RNA genome of the retroviral vector particle or areincapable of encoding functional auxiliary proteins. In a furtherembodiment, at least two or more, three or more, four or more, or all ofthe auxiliary genes vpr, vif, vpu, tat and nef, or analogous auxiliarygenes, are disrupted such that said auxiliary genes are removed from theRNA genome of the retroviral vector particle or are incapable ofencoding functional auxiliary proteins. Removal of the functionalauxiliary gene may not require removal of the whole gene; removal of apart of the gene or disruption of the gene will be sufficient.

It will be understood that the nucleic acid sequences encoding thereplication defective retroviral vector particle may be the same as, orderived from, the wild-type genes of the retrovirus upon which theretroviral vector particle is based, i.e. the sequences may begenetically or otherwise altered versions of sequences contained in thewild-type virus. Therefore, the retroviral genes incorporated into thenucleic acid vectors or host cell genomes, may also refer tocodon-optimised versions of the wild-type genes.

Additional Components

The nucleic acid vectors of the invention may comprise furtheradditional components. These additional features may be used, forexample, to help stabilize transcripts for translation, increase thelevel of gene expression, and turn on/off gene transcription.

The retroviral vector particles produced by the invention may be used inmethods of gene therapy. Therefore, in one embodiment, the nucleic acidvector additionally comprises one or more transgenes. This transgene maybe a therapeutically active gene which encodes a gene product which maybe used to treat or ameliorate a target disease. The transgene mayencode, for example, an antisense RNA, a ribozyme, a protein (forexample a tumour suppressor protein), a toxin, an antigen (which may beused to induce antibodies or helper T-cells or cytotoxic T-cells) or anantibody (such as a single chain antibody). In one embodiment, thetransgene encodes beta globin.

Multiple copies of the transfer vector containing the transgene areexpected to result in higher retroviral vector titre, therefore in oneembodiment, the nucleic acid vector comprises multiple copies of thetransgene, such as two or more, in particular three or more, copies ofthe transgene. In some cases more than one gene product is required totreat a disease, therefore in a further embodiment, the nucleic acidvector additionally comprises two or more, such as three or more, orfour or more, different transgenes.

References herein to “transgene” refer to heterologous or foreign DNAwhich is not present or not sufficiently expressed in the mammalian hostcell in which it is introduced. This may include, for example, when atarget gene is not expressed correctly in the mammalian host cell,therefore a corrected version of the target gene is introduced as thetransgene. Therefore, the transgene may be a gene of potentialtherapeutic interest. The transgene may have been obtained from anothercell type, or another species, or prepared synthetically. Alternatively,the transgene may have been obtained from the host cell, but operablylinked to regulatory regions which are different to those present in thenative gene. Alternatively, the transgene may be a different allele orvariant of a gene present in the host cell.

The aim of gene therapy is to modify the genetic material of livingcells for therapeutic purposes, and it involves the insertion of afunctional gene into a cell to achieve a therapeutic effect. Theretroviral vector produced using the nucleic acid vectors and host cellsdescribed herein can be used to transfect target cells and induce theexpression of the gene of potential therapeutic interest. The retroviralvector can therefore be used for treatment of a mammalian subject, suchas a human subject, suffering from a condition including but not limitedto, inherited disorders, cancer, and certain viral infections.

In one embodiment, the nucleic acid vector additionally comprises atranscription regulation element. For example, any of the elementsdescribed herein may be operably linked to a promoter so that expressioncan be controlled. Promoters referred to herein may include knownpromoters, in whole or in part, which may be constitutively acting orinducible, e.g. in the presence of a regulatory protein. In oneembodiment, the nucleic acid vector additionally comprises a highefficiency promoter, such as a CMV promoter. This promoter has theadvantage of promoting a high level of expression of the elementsencoded on the non-mammalian nucleic acid vector. In a furtherembodiment, the CMV promoter comprises a sequence derived from the humancytomegalovirus strain AD169. This sequence is available at GenomeAccession No. X17403, for example from base pairs 173731 to 174404.

In one embodiment, the nucleic acid vector additionally comprises aninsulator, such as a chromatin insulator. The term “insulator” refers toa genetic sequence which blocks the interaction between promoters andenhancers. In a further embodiment, the insulator (such as a chromatininsulator) is present between each of the retroviral nucleic acidsequences. This helps to prevent promoter interference (i.e. where thepromoter from one transcription unit impairs expression of an adjacenttranscription unit) between adjacent retroviral nucleic acid sequences.It will be understood that if the insulators are present in the nucleicacid vector between each of the retroviral nucleic acid sequences, thenthese may be arranged as individual expression constructs within thenucleic acid vector. For example, each sequence encoding the retroviralnucleic acid sequences has its own promoter and/or an intron and/orpolyA signal. In one embodiment, the chromatin insulator has at least90% sequence identity, for example at least 95% sequence identity, tothe chicken (Gallus gallus) HS4 insulator sequence (for example seeGenome Accession No. U78775.2, base pairs 1 to 1205).

In one embodiment, the nucleic acid vector additionally comprises apolyA signal. The use of a polyA signal has the advantage of protectingmRNA from enzymatic degradation and aiding in translation. In a furtherembodiment, the polyA signal is obtained from or derived from SV40,Bovine Growth Hormone and/or Human Beta Globin. In one embodiment, thepolyA signal is derived from the SV40 early polyA signal (for example,see Genome Accession No. EF579804.1, base pairs 2668 to 2538 from theminus strand). In one embodiment, the polyA signal is derived from theHuman Beta Globin polyA signal (for example, see Genome Accession No.GU324922.1, base pairs 3394 to 4162).

In one embodiment, the nucleic acid vector additionally comprises anintron sequence. The use of an intron downstream of theenhancer/promoter region and upstream of the cDNA insert (i.e. thetransgene) is known to increase the level of expression of the insert.In a further embodiment, the intron sequence is a Human Beta GlobinIntron or the Rabbit Beta Globin Intron II sequence. In one embodiment,the Human Beta Globin Intron is derived from the sequence available atGenome Accession No. KM504957.1 (for example from base pairs 476 to1393). In one embodiment, the Rabbit Beta Globin Intron II is derivedfrom the sequence available at Genome Accession No. V00882.1 (forexample, from base pairs 718 to 1290).

In one embodiment, the nucleic acid vector additionally comprises awoodchuck hepatitis virus post-transcriptional regulatory element(WPRE). The presence of WPRE has been shown to enhance expression and assuch is likely to be beneficial in attaining high levels of expression.In a further embodiment, the WPRE is derived from the sequence availableat Genome Accession No. J04514.1 (for example, from base pairs 1093 to1684).

In one embodiment, the nucleic acid vector additionally comprises aninternal ribosome entry site (IRES). An IRES is a structured RNA elementthat is usually found in the 5′-untranslated region downstream of the5′-cap (which is required for the assembly of the initiation complex).The IRES is recognized by translation initiation factors, and allows forcap-independent translation. In a further embodiment, the IRES isderived from the Encephalomyocarditis virus (EMCV) genome (for example,see Genome Accession No. KF836387.1, base pairs 151 to 724).

In one embodiment, the nucleic acid vector additionally comprises aMultiple Cloning Site (MCS). An MCS is a short segment of DNA within thenucleic acid vector which contains multiple restriction sites (forexample, 10, 15 or 20 sites). These sites usually occur only once withinthe nucleic acid vector to ensure that the endonuclease only cuts at onesite. This allows for the retroviral genes to be easily inserted usingthe appropriate endonucleases (i.e. restriction enzymes).

It will be understood by a person skilled in the art that the constructsmay be arranged in any order within the nucleic acid vector. In anexemplary embodiment, the nucleic acid vector comprises the followinginsert: a retroviral nucleic acid sequence encoding the gag and polproteins, a retroviral nucleic acid sequence encoding the env protein ora functional substitute thereof (such as VSVg) and a retroviral nucleicacid sequence encoding the auxiliary gene rev (such as a codon optimisedrev sequence) or an analogous gene thereto or a functionally analogoussystem (i.e., GagPol-Env-Rev-remaining BAC sequence (“BAC bone”); suchas: GagPol-(wild-type)VSVg-(codon-optimised)Rev-pSMARTBAC). In a furtherembodiment, an insulator (such as a chromatin insulator) is presentbetween each of the gagpol, env and rev sequences. In a furtherembodiment, a promoter is present before each of the gagpol, env and revsequences. In a yet further embodiment, at least one copy of thetransfer vector sequence (i.e. comprising nucleic acid sequences whichencode the RNA genome of a retroviral vector particle) is present beforethe gagpol sequence.

In one embodiment, the nucleic acid vector comprises the followinginsert: an insulator (such as a chromatin insulator), a promoter (suchas a CMV promoter), an intron (such as a human beta globin intron), aretroviral nucleic acid sequence encoding the gag and pol proteins, aretroviral nucleic acid encoding RRE, a polyA signal (such as a humanbeta globin polyA signal), an insulator (such as a chromatin insulator),a promoter (such as a CMV promoter), an intron (such as a human betaglobin intron), a retroviral nucleic acid sequence encoding the envprotein or a functional substitute thereof (such as VSVg), a polyAsignal (such as a human beta globin polyA signal), an insulator (such asa chromatin insulator), a promoter (such as a CMV promoter), aretroviral nucleic acid sequence encoding the auxiliary gene rev or ananalogous gene thereto or a functionally analogous system, a polyAsignal (such as a human beta globin polyA signal), an insulator (such asa chromatin insulator), a promoter (such as a CMV promoter), an intron(such as a rabbit beta globin intron), a polyA signal and a multiplecloning site. It will be understood that further sequences may beincluded with and/or within this insert.

The nucleic acid sequences may be introduced into the nucleic acidvector sequentially. This allows for selection after each integration toensure that all of the required nucleic acid sequences are successfullyintegrated into the nucleic acid vector. Alternatively, at least two ormore of the nucleic acid sequences are introduced into the nucleic acidvector simultaneously.

It will be understood that the additional genes described herein may beintroduced into the nucleic acid vector by standard molecular cloningtechniques known in the art, for example using restriction endonucleasesand ligation techniques. Furthermore, the nucleic acid vector, inparticular BACs, PACs, fosmids and/or cosmids, may be introduced intobacterial host cells (such as E. coli cells, in particular the E. colistrain DH10B) by standard techniques, such as electroporation.

Uses

According to a further aspect of the invention, there is provided thenucleic acid vector as defined herein for use in a method of producingretroviral vector particles. As described herein, the present inventionprovides multiple advantages for using the described nucleic acidvectors in transient transfection methods, mainly by reducing the 4plasmid transfection system into a single nucleic acid vector therebyreducing the amount of material used.

Methods

According to a further aspect of the invention, there is provided amethod of producing a replication defective retroviral vector particle,comprising:

-   -   (a) introducing the nucleic acid vector as defined herein into a        culture of mammalian host cells; and    -   (b) culturing the mammalian host cells under conditions in which        the replication defective retroviral vector particle is        produced.

The advantage of including all of the retroviral genes on a largenucleic acid vector is that they can be prepared in microbial cells(such as bacterial or yeast cells) first, which are much easier tohandle and manipulate, before being introduced into mammalian cells in asingle step.

In one embodiment, the host cell is a mammalian cell. In a furtherembodiment, the mammalian cell is selected from a HEK 293 cell, HEK 6Ecell, CHO cell, Jurkat cell, KS62 cell, PerC6 cell, HeLa cell, HOS cell,H9 cell or a derivative or functional equivalent thereof. In a yetfurther embodiment, the mammalian host cell is a HEK 293 cell, orderived from a HEK 293 cell. Such cells could be adherent cell lines(i.e. they grow in a single layer attached to a surface) or suspensionadapted/non-adherent cell lines (i.e. they grow in suspension in aculture medium). In a yet further embodiment, the HEK 293 cell is a HEK293T cell or a HEK 6E cell. The term “HEK 293 cell” refers to the HumanEmbryonic Kidney 293 cell line which is commonly used in biotechnology.In particular, HEK 293T cells are commonly used for the production ofvarious retroviral vectors. Other examples of suitable commerciallyavailable cell lines include T REX™ (Life Technologies) cell lines.

The host cells transduced using the methods defined herein may be usedto produce a high titre of retroviral vector.

References herein to the term “high titre” refer to an effective amountof retroviral vector or particle which is capable of transducing atarget cell, such as a patient cell. In one embodiment, a high titre isin excess of 10⁶ TU/ml without concentration (TU=transducing units).

The skilled person will be aware that introducing the nucleic acidvector into the host cell may be performed using suitable methods knownin the art, for example, lipid-mediated transfection (lipofection),microinjection, cell (such as microcell) fusion, electroporation,chemical-based transfection methods or microprojectile bombardment. Itwill be understood that the choice of method to use for introducing thenucleic acid vector can be chosen depending upon the type of mammalianhost cell used. In one embodiment, introduction step (a) is performedusing lipofection, electroporation or a chemical-based transfectionmethod. In a further embodiment, the nucleic acid vector is introducedinto the host cell by lipofection. In an alternative embodiment, thenucleic acid vector is introduced into the host cell by a chemical-basedtransfection method, such as calcium phosphate treatment. Calciumphosphate treatments are commercially available, for example fromPromega.

It will be understood by the skilled person that the conditions used inthe method described herein will be dependent upon the host cell used.Typical conditions, for example the culture medium or temperature to beused, are well known in the art (e.g. see Kutner et al. (2009) NatureProtocols 4(4); 495-505). In one embodiment, culturing step (b) isperformed by incubating the mammalian host cell under humidifiedconditions. In a further embodiment, the humidified conditions compriseincubating the transfected cells at 37° C. at 5% CO₂. In one embodiment,culturing step (b) is performed using a culture medium selected from:Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) fetalbovine serum (FBS) or serum-free UltraCULTURE™ medium (Lonza, Cat. No.12-725F) or FreeStyle™ Expression medium (Thermo fisher, Cat. No. 12338018).

In one embodiment, the method additionally comprises isolating thereplication defective retroviral vector particle. For example, in oneembodiment the isolating is performed by using a filter. In a furtherembodiment, the filter is a low-protein binding membrane (e.g. a 0.22 μmlow-protein binding membrane or a 0.45 μm low-protein binding membrane),such as polyvinylidene fluoride (PVDF) or polyethersulfone (PES)artificial membranes.

In one embodiment, the replication defective retroviral vector particlesare isolated no longer than 72 hours after introduction step (a). In afurther embodiment, the replication defective retroviral vectorparticles are isolated between 48 and 72 hours after introduction step(a), for example at 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 hours.

Once isolated, the retroviral vector particles may be concentrated forin vivo applications. Concentration methods include, for example,ultracentrifugation, precipitation or anion exchange chromatography.Ultracentrifugation is useful as a rapid method for retroviral vectorconcentration at a small scale. Alternatively, anion exchangechromatography (for example using Mustang Q anion exchange membranecartridges) or precipitation (for example using PEG 6000) areparticularly useful for processing large volumes of lentiviral vectorsupernatants.

According to a further aspect of the invention, there is provided areplication defective retroviral vector particle obtained by the methoddefined herein.

The invention will now be described in further detail with reference tothe following, non-limiting Examples.

EXAMPLES Example 1: Construct Guide

FIG. 1 shows a stepwise guide to the construction ofBACpack-WTGP-277delU5 and BACpack-SYNGP-277delU5. Owing to thecompatible ends of an XbaI and NheI digest, the lentiviral packaginggenes were progressively loaded into the pSmart BAC vector. At the pointof GagPol addition, 2 constructs were made containing either Wild typeGagPol (WTGP) or a codon optimised GagPol (SYNGP). These were given thenomenclature of BACpack-WTGP and of BACpack-SYNGP respectively. Thetransfer vector was then loaded onto both of these constructs and sogenerating BACpackWTGP-277delU5 and BACpackSYNGP-277delU5.

Example 2: Proof of Principle Experiment Using BAC Construct

10⁶ HEK293T cells were seeded in a 6 well plate. The following day, theadhered cells were transfected using PEI to manufacturer's instruction.Cells were either transfected with a total 4 μg of Wild Type (WT)lentiviral packaging constructs consisting of pMDL.gp (GagPol), pMD.G(VSVg), pK-Rev (Rev) and pCCL.277 (GFP Transfer vector) or 2 μg BACpack(a single BAC construct containing GagPol, VSVg and Rev) plus 2 μg ofthe eGFP transfer vector on a separate plasmid.

48 hours post transfection, the supernatant was harvested, filteredthrough a 0.22 μm filter and stored at −80° C. for a minimum of 4 hours.HEK293T cells were seeded for transduction at 10⁵ cells per well in a 24well plate. The following day viral supernatant was applied to the cellsin serial dilutions with Polybrene at a final concentration of 8 μg/ml.3 days post transduction the cells were harvested by trypsin treatmentand analysed for GFP by FACS. Viral titre was calculated as TransductionUnits (TU)/mL using the following equation:

(GFP positive cells/100)×dilution factor×number of cells transduced.

Viral titres are compared on the barchart (FIG. 2). All incubations werecarried out at 37° C. and 5% CO₂. Media used was DMEM supplemented withFBS to 10% and 1 μg/ml Doxycycline in the BACpack+Transfer sample.

Observations:

In this Example, the ability of the BACpack construct, consisting ofGagPol, VSVg and Rev expression cassettes was compared to the standard 3plasmid packaging system where Gag Pol, VSVg and Rev are deliveredseparately. In both cases, the transfer vector was delivered alongsidein a separate plasmid in order to complete the essential components forthe viral vector.

In this instance, the BACpack plus transfer vector was capable ofachieving an unconcentrated supernatant viral titre of 2.2×10⁷ TU/ml,compared to a titre of 5×10⁷ TU/ml when using the 4 separate plasmidlentivirus system. Although a lower titre was observed using theBACpack, this assay was performed pre-optimisation and a greater titremay be achieved post-optimisation.

From this proof of principal assay, it can be concluded that the BACpackis capable of packaging of the transfer vector at a viral titrecomparable to that of the separate packaging plasmid system in atransient transfection.

Example 3: Transient Transfection of the BACpack in Adherent HEK293TCells

In order to confirm the ability of the two BACpack-277delU5 constructsto produce lentiviral vector in a transient transfection system theadherent cell line, HEK293T, routinely used to produce lentiviral vectorby transient transfection, were transfected with either the current 4packaging plasmid system, BACpackWTGP-277delU5 or BACpackSYNGP-277delU5.The two BACpack-277delU5 constructs were either induced to assesswhether gene expression could result in lentiviral vector production orleft uninduced to test the efficiency of the Tet Repressor system.

The results in FIG. 3 show the titre in transduction units (TU)/mL, ofthe lentiviral vector supernatant harvested from each transfectioncondition. It can be seen from the titration results that cellstransfected with either BACpackWTGP-277delU5 or BACpackSYNGP-277delU5and induced with 1 ug/ml Doxycycline (+Dox) produced comparableconcentrations of lentiviral vector as the current 4 plasmid system. Inaddition, the uninduced conditions demonstrated a greatly reducedability to produce lentiviral vector compared to induced, and althoughthis production was greater than the un-transfected control background,this is not seen to be a disadvantage in a transient system.

These results suggests that the single BAC vector containing all of thepackaging genes necessary for lentiviral production could replace thecurrent 4 plasmid system.

It will be understood that the embodiments described herein may beapplied to all aspects of the invention. Furthermore, all publications,including but not limited to patents and patent applications, cited inthis specification are herein incorporated by reference as though fullyset forth.

1. A nucleic acid vector selected from: a yeast artificial chromosome(YAC), a P1-derived artificial chromosome (PAC), a fosmid, or a cosmid,characterized in that said vector comprises retroviral nucleic acidsequences encoding: gag and pol proteins, and an env protein or afunctional substitute thereof, wherein each of the retroviral nucleicacid sequences are arranged as individual expression constructs withinthe vector.
 2. The nucleic acid vector of claim 1, which additionallycomprises nucleic acid sequences which encode the RNA genome of aretroviral vector particle.
 3. The nucleic acid vector of claim 1, whichadditionally comprises the auxiliary gene rev or an analogous genethereto or a functionally analogous system.
 4. The nucleic acid vectorof claim 1, wherein the retroviral nucleic acid sequences are obtainedfrom a retrovirus selected from lentivirus, alpha-retrovirus,gammaretrovirus or foamy-retrovirus.
 5. The nucleic acid vector of claim4, wherein the retroviral nucleic acid sequences are obtained from alentivirus selected from the group consisting of HIV-1, HIV-2, SIV, FIV,EIAV and Visna.
 6. The nucleic acid vector of claim 5, wherein theretroviral nucleic acid sequences are obtained from HIV-1.
 7. Thenucleic acid vector of claim 1, wherein the env protein or a functionalsubstitute thereof is obtained from Vesicular stomatitis virus.
 8. Thenucleic acid vector of claim 1, which additionally comprises atranscription regulation element.
 9. The nucleic acid vector of claim 8,wherein the transcription regulation element is a CMV promoter.
 10. Thenucleic acid vector of claim 1, which additionally comprises aninsulator.
 11. The nucleic acid vector of claim 10, wherein an insulatoris present between each of the retroviral nucleic acid sequences. 12.The nucleic acid vector of claim 1, which additionally comprises one ormore transgenes.
 13. The nucleic acid vector of claim 1, whichadditionally comprises an Internal Ribosome Entry Site (IRES).
 14. Thenucleic acid vector of claim 1, which additionally comprises a polyAsignal.
 15. The nucleic acid vector of claim 1, which additionallycomprises an intron sequence.
 16. The nucleic acid vector of claim 1,which additionally comprises a Multiple Cloning Site (MCS).
 17. A methodof producing a replication defective retroviral vector particle,comprising: (a) introducing into a culture of mammalian host cells, anucleic acid vector selected from: a yeast artificial chromosome (YAC),a P1-derived artificial chromosome (PAC), a fosmid, or a cosmid,characterized in that said vector comprises retroviral nucleic acidsequences encoding: gag and pol proteins, and an env protein or afunctional substitute thereof wherein each of the retroviral nucleicacid sequences are arranged as individual expression constructs withinthe vector; and (b) culturing the mammalian host cells under conditionsin which the replication defective retroviral vector particle isproduced.
 18. The method of claim 17, wherein the mammalian host cell isa HEK 293 cell.
 19. The method of claim 17, wherein introduction step(a) is performed using lipofection, electroporation or a chemical-basedtransfection method, such as calcium phosphate treatment.
 20. The methodof claim 17, wherein culturing step (b) is performed by incubating themammalian host cell under humidified conditions.
 21. The method of claim17, additionally comprising isolating the replication defectiveretroviral vector particle.
 22. The method of claim 21, wherein theisolating is performed by using a filter, such as a low-protein bindingmembrane.
 23. The method of claim 21, wherein the replication defectiveretroviral vector particles are isolated no longer than 72 hours afterintroduction step (a).
 24. The method of claim 23, wherein thereplication defective retroviral particles are isolated between 48 and72 hours after introduction step (a).